Treatment of hmgb1-mediated inflammation

ABSTRACT

Methods of treating HMGB1-mediated inflammation by administering a therapeutically effective amount of an MD2-antagonist to a subject in need thereof are described. The novel MD2 antagonist tetrapeptide P5779 is also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/090,934, filed Dec. 12, 2014, and U.S. Provisional PatentApplication Ser. No. 62/222,486, filed Sep. 232015, both of which areincorporated herein by reference.

GOVERNMENT FUNDING

This invention was made with Government support under grant numbersRO1GM62508, RO1GM098446, 5P50GM053789, RO1GM107876 and RO1AT005076awarded by the National Institutes of Health and the National CancerInstitute. The Government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 14, 2015, isnamed HMGB1 NSLJ1-024062_ST25 and is 4,096 bytes in size.

BACKGROUND

Following infection or injury, the immediate host inflammatory responseis mediated by receptors on innate immune cells that can efficientlyrecognize pathogen- or damage-associated molecular patterns (PAMPs orDAMPs). For instance, the mammalian response to bacterial endotoxin(lipopolysaccharide, LPS) is mediated by the LPS-binding protein (LBP),CD14, MD2, and TLR4. Upon capturing LPS, LBP transfers it to CD14 andMD2, which then delivers LPS to the signaling, high-affinitytransmembrane Toll-like receptor 4 (TLR4). Nagai et al., Nat Immunol3:667-672 (2002). The engagement of LPS with TLR4 triggers thesequential release of “early” (e.g., TNF, IL-1, IFN-β) and “late”pro-inflammatory mediators (e.g., HMGB1) Wang et al., Science285:248-251 (1999).

As a ubiquitous nuclear protein, HMGB1 can be passively released fromdamaged cells following sterile tissue injury due toischemia/reperfusion (Tsung et al., J Exp Med 201:1135-1143 (2005)) orchemical toxicity. Antoine et al., Hepatology 58:777-787 (2013). HMGB1can signal through a family of receptors including RAGE, TLR4, andcluster of differentiation 24 (CD24)/Siglec-10, thereby functioning as aDAMP that alerts, recruits and activates innate immune cells to producea wide range of cytokines and chemokines. Thus, seemingly unrelatedconditions such as infection and sterile injury can converge on a commonprocess: inflammation, which is orchestrated by HMGB1 actively secretedfrom innate immune cells or passively released from damaged tissues.Andersson, U. and Tracey, K. J., Annu Rev Immunol 29:139-162 (2011).Extracellular HMGB1 has been established as a pathogenic mediator ofboth infection- and injury-elicited inflammatory diseases. Yang et al.,J Leukoc Biol 93:865-873 (2013).

HMGB1 is a redox-sensitive protein as it contains three conservedcysteine residues at position 23, 45 and 106. The redox status of thecysteines dictates its extracellular chemokine or cytokine-inducingproperties. Specifically, HMGB1 with all cysteine residues reduced(fully reduced HMGB1) binds to CXCL12 and stimulates immune cellinfiltration via the CXCR4 receptor in a synergistic fashion. Partiallyoxidized HMGB1, with a Cys23-Cys45 disulfide bond and a reduced Cys106(disulfide HMGB1), activates immune cells to producecytokines/chemokines via the TLR4 receptor. Once all cysteines areterminally oxidized (sulfonyl HMGB1), HMGB1 is devoid of chemotactic andcytokine activities. Previously we showed that HMGB1 inducesinflammatory responses via the TLR4/MD2 signaling pathway, and that theinteraction with TLR4/MD2 requires a specific HMGB1 redox form with adistinct atomic structure of thiol-cysteine 106. Yang et al., Mol Med18:250-259 (2012). Ample evidence suggests that HMGB1, when activelysecreted by activated immune cells or passively released from dyingcells, is a mixture of several isoforms with distinct post-translationalmodifications. Paradoxically, it is unknown how the immune system usesthe TLR4/MD2 receptor system to distinguish between different isoformsof HMGB1, specifically recognizing the disulfide HMGB1 molecule to theexclusion of other isoforms.

A type HMGB1-mediated inflammation that is of particular interest isthat caused by Influenza. Influenza continues to evolve with newantigenic variants emerging annually, as exemplified by the last severalinfluenza seasons in which the recommended vaccine was considerably lessefficacious than predicted. Therefore, there remains a pressing need todevelop alternatives to the annual influenza vaccines and anti-viralagents currently used to mitigate the effects of influenza infection.Influenza virus is sensed by multiple PRRs, including TLR3, TLR7, TLR8,TLR10, and the intra-cytosolic sensor, RIG-I, although TLR8 and TLR10are not functional in mice. CD14 is required for influenza-inducedcytokine production by mouse macrophages, independent of TLR2 and TLR4.In addition, MyD88^(−/−) and MyD88/TRIF double deficient mice show adramatic reduction of pulmonary cytokine production when compared to WTmice, indicating the important role of these TLR signaling pathways indisease.

Imai et al. proposed that chemical or microbial insults triggerNADPH-dependent reactive oxygen species that generate a host-derivedoxidized phospholipid, oxidized1-palmitoyl-2-arachidonoyl-phosphaticylcholine (OxPAPC), in lungs. Imaiet al., Cell 133, 235-249 (2008). They concluded that regardless of theinitial sensing involved in pathogen recognition, OxPAPC initiates acommon TLR4-, TRIF-, and IL-6-dependent pathway in macrophages thatleads to ALI. We showed that treatment of influenza-infected mice withEritoran, the most potent, synthetic lipid A analog known (Lien et al.,J. Biol. Chem. 276, 1873-1880 (2001)), blocked influenza-inducedlethality and ALI. When administered daily to WT mice for 5 days,starting on days 2, 4, or 6 post-infection, Eritoran treatmentsignificantly improved survival and clinical symptoms, while decreasingALI, OxPAPC accumulation, the cytokine storm, and systemic inflammation.Shirey et al., Nature 497, 498-502 (2013).

SUMMARY OF THE INVENTION

The present invention provides methods of treating or preventingHMGB1-mediated inflammation in a subject, by administering atherapeutically effective amount of an MD2-antagonist to a subject inneed thereof. In some embodiments, the HMGB1 is the HMGB1 disulfideisoform. When treating HMGB1-mediated inflammation, the MD2-antagonistis administered after the onset of infection in some embodiments. Insome embodiments, the MD2-antagonist is administered in apharmaceutically acceptable carrier.

HMGB1-mediated inflammation can result from infection or sterile injury.In some embodiments, the HMGB1-mediated inflammation is caused by viralinfection, such as influenza infection. In some embodiments, whentreating HMGB1 inflammation that is caused by viral infection, themethod further comprises administering an anviral agent (e.g.,oseltamivir) to the subject. In other embodiments, the HMGB1-mediatedinflammation is caused by bacterial infection. In further embodiments,the HMGB1-mediated inflammation is caused by sterile injury, such asacetaminophen toxicity.

Another aspect of the invention provides a composition comprising theMD2 antagonist having the amino acid sequence FSSE. In some embodiments,the MD2 antagonist composition further comprises a pharmaceuticallyacceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

The present invention may be more readily understood by reference to thefollowing figures, wherein:

FIGS. 1A-1D provide graphs showing disulfide HMGB1 binds to MD2. (FIG.1A) TNF release was measured from RAW 264.7 cells stimulated withvarious isoforms of HMGB1 as indicated (1 μg/ml, 16 h). *: P<0.05 vs.disulfide HMGB1. N=3-5 experiments. (FIG. 1B) Surface plasmon resonance(SPR, BIAcore) analysis was performed to assess HMGB1 binding to MD2 orTLR4 (coated on the chip). Upper row: HMGB1 binding to human MD2 wastested at different HMGB1 concentrations (12.5, 25, 50 and 100 nM) withan apparent Kd of 12 nM (left graph). Human MD2 (12.5, 25, 50 and 100nM) binding to HMGB1 (coated on the chip, middle graph); disulfide HMGB1(100 nM) was tested binding to TLR4 (coated on the chip, right graph).Lower row: Non-cytokine-inducing HMGB1 (C106A, Hg-HMGB1, 1 μM) weretested binding to MD2 (coated on the chip, left graph). HMGB1 isoformswere tested for binding to MD2 (coated on the chip, right graph). Dataare presented as response units (RU) or relative RU over time (seconds)and representative of three experiments. (FIG. 1C) Mixture of CBP-taggedHMGB1 or CBP alone with supernatant of yeast Sf9 cells expressing MD2was immune-precipitated with calmodulin beads (immune-precipitation,IP), and immunoblotted (IB) with anti-human MD2 or CBP antibodies.Recombinant MD2 protein was included as positive control (right lane).Data shown are representative of 3 repeats. (FIG. 1D) SPR analysis ofHMGB1 binding to human MD2 (coated on the chip) was performed in thepresence of monoclonal anti-HMGB1 mAb (left graph) or irrelevant mouseIgG (right graph) as shown. Data are representative of 3 repeats.

FIGS. 2A-2C provide graphs and images showing MD2 is indispensable forHMGB1-dependent TLR4 signaling. (FIG. 2A) Upper panel: knockdown of MD2(siRNA) was performed on RAW 264.7 cells. MD2 and NF-κB levels (p65)were assessed by western blotting. The level of NF-κB (p65) protein wasnormalized relative to level of ß-actin (ratio) by densitometry, andexpressed as the fold change over un-stimulated cells. Lower panel:HMGB1-induced TNF release from RAW 264.7 cells with MD2 knockdown (openbars) or control siRNA (solid bars). *: P<0.05 vs. control siRNA group.N=4-5 experiments. (FIG. 2B) Left panel:HMGB1 (2 μg/ml) or ultrapure LPS(200 ng/ml) were used to stimulate primary peritoneal macrophages fromwild type (WT) or MD2 KO mice for 16 hours, and NF-κB (p50 and p65)protein levels in nuclear extracts were assessed by western blotting(upper left panels). NF-κB activation is expressed as of p50 or p65relative to ß-actin and calculated as the fold change over un-stimulatedcells (lower left graphs). Right panel: Mouse macrophages werestimulated with HMGB1 and cytokine released was measured using mousecytokine Ab array (G-CSF, IL-12p40, IL-6, TNF, RANTES, MCP-1, sTNFR1;upper right panel) or ELISA (for TNF, lower right graph). *: P<0.05 vs.WT group. N=5 separate experiments. (FIG. 2C) WT or MD2 KO mice werechallenged with APAP in a liver injury model, and were euthanized 24 hlater to measure serum levels of liver enzymes (GLDH, ALT, and AST; leftcolumn graphs) and cytokines (HMGB1, TNF and IL-6; right column graphs).*: P<0.05 vs. WT APAP group. N=5-13 mice per group. Representative H&Estaining of liver tissues from these mice are shown. N=5-8 mice pergroup (magnification, ×200; the arrow indicates necrosis region; middlepanel). Scale bar=100 μm Animal survival after receiving lethal dose ofAPAP in WT and MD2 KO mice was assessed (percent survival). N=15 miceper group. *: P<0.05 vs. wild type (right panel graph).

FIGS. 3A and 3B provide graphs showing monoclonal anti-HMGB1 antibodyadministration ameliorates APAP-induced liver injury in mice. (FIG. 3A)Mice received an APAP injection (i.p.) followed by treatment with ananti-HMGB1 antibody or control IgG injection (i.p., see Methods). Animalsurvival (% survival) was assessed. N=20 mice/group. *: P<0.05 vs. IgGgroup. (FIG. 3B) Serum levels of liver enzyme (ALT) and cytokines (TNFand IL-6) at 24 h post-APAP were measured in mice receiving treatment ofanti-HMGB1 Ab or control IgG (see Methods). N=10 mice/group. *: P<0.05vs. IgG group.

FIGS. 4A-4D provide graphs and images showing the results of screeningfor HMGB1 inhibitors. (FIG. 4A) SPR analysis was performed to test theinteraction of MD2 (coated on the chip) with P5779 (FSSE) and otherpeptides (100 nM). Kd values are shown. Data are representative of 3experiments. (FIG. 4B) Primary human macrophages were stimulated invitro with HMGB1 (1 μg/ml) plus different peptides (50 μg/ml) for 16 h,and TNF release was measured by ELISA. N=4-5 experiments. *: P<0.05 vs.HMGB1 alone. (FIG. 4C) SPR analysis was performed to measure binding ofP5779 (12.5, 25, 50 and 100 nM), or scrambled control (ctrl) peptide(100 nM), to human MD2 (Kd=0.65 μM for P5779), HMGB1 or TLR4 (coated onthe chip). Data are representative of three experiments.(FIG. 4D)Schematic illustration showing molecular docking of MD2 with tetramerpeptides FSSE (left) and SFSE (right). The brown area represents thesurface of the peptide binding pocket of MD2 and the green area denotesthe TLR4 protein surface. The lower panel shows hydrogen bonds and vander Waals interactions. P5779, with a stronger van der waals interactionthan control is fully extended into the hydrophobic pocket of MD2 andforms an additional hydrogen bond with Tyr102 of MD2.

FIGS. 5A-5E provides graphs and images showing the development of atetramer peptide (P5779) as an MD2-binding HMGB1-specific inhibitor.(FIG. 5A) On SPR analysis, HMGB1 was coated on the chip and MD2 (1 μM)was flowed over as analyte, plus different amounts of P5779 as shown.Inhibition of HMGB1 binding to MD2 by P5779 (IC₅₀=29 nM) was assessed(upper graph). In the reverse experiment, human MD2 was coated on thechip and HMGB1 (1 μM) plus different amounts of P5779 were added asanalytes. HMGB1 binding to MD2 was inhibited by P5779 (IC₅₀=2 nM) (lowergraph). Data are representative from 3 separate experiments. (FIG. 5B)Human primary macrophages, isolated from human blood, were stimulatedwith HMGB1 (1 μg/ml), or other stimuli (Poly I:C, S100A12, LPS, PGN andCpGDNA) in vitro, plus increasing amounts of P5779 (or scrambled controlpeptide) for 16 hours. TNF released was measured by ELISA. N=4-5experiments. *: P<0.05 vs. HMGB1 plus control peptide (ctrl). (FIG. 5C)Thioglycollate-elicited peritoneal mouse macrophages were stimulated invitro with HMGB1 (1 μg/ml) plus P5779 or control peptide (ctrl, 50μg/ml) for 16 h, and extracellular levels of various cytokines wereanalyzed by mouse Cytokine Ab Array (left panels). Data arerepresentative of 3-4 experiments, each performed in duplicate andexpressed as fold increase over unstimulated cells using densitometry(−HMGB1) (right table). *: P<0.05 vs. +HMGB1 group. (FIG. 5D) Primaryhuman macrophages, isolated from blood, were stimulated in vitro withLPS (2 ng/ml) for 16 h in the absence or presence of P5779 (50 μg/ml) orcontrol peptide (ctrl), and extracellular levels of various cytokineswere analyzed by human cytokine Ab array (left panels). Data arerepresentative of 3 repeats. (FIG. 5E) Male C57BL/6 mice received an LPSinjection (8 mg/kg, i.p.) plus P5779 or control peptide (ctrl) (500μg/mouse, i.p.). Animals were euthanized 90 minutes later. Serum TNF andIL-6 levels were measured by ELISAs. N=5 mice per group.

FIGS. 6A-6C provide graphs and images showing treatment with the HMGB1inhibitor, P5779, ameliorates APAP-mediated toxicity,ischemia/reperfusion injury and sepsis mortality in vivo. (FIG. 6A)C57BL/6 mice received an APAP injection (i.p. Methods) and wereadministered with P5779 (at doses indicated) or control peptide (ctrl,500 μg/mouse; i.p.). Mice were euthanized at 24 h post-APAP and serumenzyme levels (ASL and ALT) and cytokine levels (TNF) were measured byELISAs (left upper graphs). N=6-10 mice per group. In survivalexperiments, mice received an APAP injection (i.p.) and wereadministered with P5779 or control peptide (ctrl, i.p. see Methods).Survival was monitored for 2 weeks (percent survival). N=30 mice/group(left lower graph). Right Panels: Representative H&E images of livertissue sections are shown for normal (untreated) or APAP-injected micereceiving P5779 or control peptides. Clinical scores were assessed andshown on the right. Liver necrosis is demonstrated by arrows(magnification, ×200). Scale bar=100 μm. N=6-10 mice/group. *: P<0.05vs. control peptide group. (FIG. 6B) P5779 or control peptide wasadministered (500 μg/mouse, i.p.) at the time of ischemia/reperfusion(I/R) surgery and mice were euthanized 6 h later to measure serum levelsof ALT and AST (left column graphs), and to evaluate histological liverinjury. *: P<0.05 vs. I/R group. N=5-7 mice/group. Representative H&Eliver tissue sections are shown (right panels, neutrophil infiltration:arrow; magnification, ×200). Scale bar=100 μm. N=3-5 mice per group.(FIG. 6C) Mice received CLP surgery, and P5779 or control peptide (Ctrl)was administered i.p. at the doses indicated. Animal survival wasmonitored for 2 weeks (% survival). N=20 mice/group. *: P<0.05 vs.control peptide group.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have described a method of treating HMGB1-mediatedinflammation in a subject by administering a therapeutically effectiveamount of an MD2-antagonist to a subject in need thereof.

Definitions

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. As used in the description of the invention andthe appended claims, the singular forms “a”, “an”, and “the” areinclusive of their plural forms, unless contraindicated by the contextsurrounding such.

Treat”, “treating”, and “treatment”, etc., as used herein, refer to anyaction providing a benefit to a subject afflicted with HMGB1-mediatedinflammation, including improvement in the condition through lesseningor suppression of at least one symptom, delay in progression of thedisease or condition, etc.

The terms “polypeptide” and “peptide” are used interchangeably herein torefer to a polymer of amino acids. These terms do not connote a specificlength of a polymer of amino acids. Thus, for example, the termsoligopeptide, protein, and enzyme are included within the definition ofpolypeptide or peptide, whether produced using recombinant techniques,chemical or enzymatic synthesis, or naturally occurring. This term alsoincludes polypeptides that have been modified or derivatized, such as byglycosylation, acetylation, phosphorylation, and the like.

“Amino acid” is used herein to refer to a chemical compound with thegeneral formula: NH₂—CRH—COOH, where R, the side chain, is H or anorganic group. Where R is organic, R can vary and is either polar ornonpolar (i.e., hydrophobic). The amino acids of this invention can benaturally occurring or synthetic (often referred to asnonproteinogenic).

The following abbreviations are used throughout the application:A=Ala=Alanine, T=Thr=Threonine, V=Val=Valine, C=Cys=Cysteine,L=Leu=Leucine, Y=Tyr=Tyrosine, I=Ile=Isoleucine, N=Asn=Asparagine,P=Pro=Proline, Q=Gln=Glutamine, F=Phe=Phenylalanine, D=Asp=AsparticAcid, W=Trp=Tryptophan, E=Glu=Glutamic Acid, M=Met=Methionine,K=Lys=Lysine, G=Gly=Glycine, R=Arg=Arginine, S=Ser=Serine,H=His=Histidine.

“Pharmaceutically acceptable” as used herein means that the compound orcomposition is suitable for administration to a subject for the methodsdescribed herein, without unduly deleterious side effects in light ofthe severity of the disease and necessity of the treatment.

The term “therapeutically effective” is intended to qualify the amountof each agent which will achieve the goal of decreasing disease severitywhile avoiding adverse side effects such as those typically associatedwith alternative therapies. The therapeutically effective amount may beadministered in one or more doses. An effective dose, on the other hand,is an amount sufficient to provide a certain effect, such as enzymeinhibition, but may or may not be therapeutically effective.

Methods of Treating HMGB1-Mediated Inflammation

In one aspect, the present invention provides methods of treatingHMGB1-mediated inflammation in a subject, by administering atherapeutically effective amount of an MD2-antagonist to a subject inneed thereof. High mobility group box 1 protein (HMGB1) is a mediator ofboth infection- and injury-elicited inflammatory diseases andconditions. Activated macrophages and monocytes secrete HMGB1 as acytokine mediator of Inflammation. HMGB1 is also known as high-mobilitygroup protein 1 (HMG-1) and amphoterin, and is encoded by the HMGB1gene. Several different isoforms of HMGB1 with distinctpost-translational modifications are involved in inflammation.Accordingly, in some embodiments, the HMGB1-mediated disease is mediatedby one or more HMGB1 isoforms. For example, in some embodiments, theHMGB1 is the HMGB1 disulfide isoform.

HMGB1-mediated inflammation, as defined herein, is a disease orcondition in HMGB1 plays a significant role in the pathology of thedisease. As further described herein, HMGB1 is secreted by immune cellssuch as macrophages, monocytes and dendritic cells as a cytokinemediator of Inflammation. HMGB1 induces inflammatory responses via theTLR4/MD2 signaling pathway as a result of binding to TLR4, whichmediates HMGB1-dependent activation of macrophage cytokine release. As aresult, HMGB1 is involved in both sterile and infectious inflammatoryresponses.

Inflammatory disease includes a wide variety of disorders characterizedby pathological inflammation of tissue Immunoactivation, which evolvedas a system of host defense against pathogens, can become dysregulatedand promote the pathogenesis of diverse diseases with both known andunknown etiologies. Immunoactivation and associated inflammation seemsto be a “common denominator” or general mechanism of pathogenesis andmay explain the association and similarities in pathology amongotherwise unrelated human diseases. Margolis, L., Am J Med. 128(6):562-6(2015). Examples of inflammatory disease include Acne vulgaris, Sepsis,Asthma, Celiac disease, Chronic prostatitis, Glomerulonephritis,Inflammatory bowel diseases, Pelvic inflammatory disease,Ischemia-Reperfusion injury, Rheumatoid arthritis, Sarcoidosis,Vasculitis, house dust mite-induced airway inflammation, andInterstitial cystitis. HMGB1 plays an important role in theseinflammatory responses for all of these diseases. See Yang et al., MolMed 21:S6-S12 (2015); Kang et al. Mol Aspects Med. 1-116 (2014);Andersson et al., Annual Rev Immunol 29:139-62 (2011).

In some embodiments, the HMGB1-mediated inflammation is due infection.Inflammation plays an important role in protection against infection,and involves eliminating the initial cause of cell injury, clearing outnecrotic cells and tissues damaged from the original insult and theinflammatory process, and to initiating tissue repair. However, in somecases, infection can induce excessive and potentially dangerousinflammation. For example, viral triggering of cytokine-mediated lunginflammation can play a significant role in virulence of infection.Likewise, endotoxins resulting from bacterial infection can causesepsis, which is a form of HMGB1-mediated inflammation.

HMGB1-mediated inflammation can result from various types of infection.For example, in some embodiments, HMGB1-mediated inflammation is causedby viral infection. Examples of viruses that can cause pathologicalinflammation include, for example, dengue virus, Hepatitis B virus (Caoet al, Sci Rep. 5:14240-5, 2015), influenza A virus (H1N1) (Nosaka etal., Critical Care, 19:249-258, 2015), chicken infectious anemia virus(Sawant et al., Vaccine. 33:333-40, 2015), human papillomavirus (Weng etal., Mol Med Rep. 10:1765-71, 2014). In some embodiments, theHMGB1-mediated inflammation is caused by influenza infection, whichfrequently causes pathological inflammation.

In other embodiments, the HMGB1-mediated inflammation is caused bybacterial infection. Pathological inflammation can occur as a result ofinfection by a wide variety of different types of bacteria. Examples ofbacteria that can trigger a pathological inflammation response includeMycobacterium tuberculosis, bacterium Burkholderia pseudomallei,bacterium Francisella tularensis (Kang et al., Mol Aspects Med. 1-116,2014; Laws et al., Internation J of Infect Dis 40:1-8, 2015. D'Elia R Vet al., Antimicrob Agents Chemother June issue PMCID: PMC3754292, 2013),Pseudomonas aeruginosa, Gram-negative pathogen-induced KeratitisMcClellan et al., J Immunol. 194:1776-1787, 2015).

Alternately, in some embodiments, HMGB1-mediated inflammation is causedby factors other than infection. Inflammation caused by factors otherthan infection is referred to herein as inflammation resulting from“sterile injury.” Sterile injury can trigger an acute inflammatoryresponse, which might be responsible for the pathogenesis of severaldiseases, including rheumatoid arthritis, lung fibrosis and acute liverfailure. Examples of sterile inflammation include acetaminophentoxicity, wound healing, rheumatoid arthritis, hemorrhagic shock,myocardial infarction, ischemia-reperfusion injury and transplantation,cerebral ischemia and injury (Kang et al., Mol Aspects Med. 1-116, 2014.Andersson et al., Annual Rev Immunol 29:139-62, 2011, Yang et al., MolMed 21:S6-S12, 2015). In some embodiments, the HMGB1-mediatedinflammation is caused by acetaminophen toxicity.

MD2-Antagonists

A preferred method of treating HMGB1-mediated inflammation in a subjectis administration of a therapeutically effective amount of a myeloiddifferentiation protein 2 (MD2) antagonist to the subject. One of theadvantages of treatment in an MD2 antagonist over many other methods oftreating inflammation is that treatment with an MD2 antagonist does notsubstantially decrease anti-microbial immune responsiveness.

An MD2 antagonist is a compound which interferes with the activity ofMD2. For example, an MD2 antagonist can interfere with the bindingbetween MD2 and another peptide, such as TLR4 or HMGB1. Hawkins et al.described Eritoran and related compounds, which are lipid-basedcompounds that can be used as MD2 antagonists. Hawkins et al., Curr TopMed Chem. 4(11):1147-71 (2004). Another MD2-antagonist, identified bythe inventors, is P5779, which is a tetrapeptide having the amino acidsequence FSSE. A further MD2-antagonist is the peptide MD2-I. Slivka etal., Chembiochem. 10(4): 645-649 (2009).

Candidate MD2 antagonists may be tested in animal models. For example,the animal model can be one for the study of inflammation. The study ofinflammation in animal models (for instance, mice) is a commonlyaccepted practice. For instance, Chen et al. discuss differences betweenhumans and murine models for evaluating sepsis. Chen et al., Surg ClinNorth Am. 94(6):1135-49 (2014). Results are typically compared betweencontrol animals treated with candidate agents and the controllittermates that did not receive treatment. Transgenic animal models arealso available and are commonly accepted as models for human disease(see, for instance, Greenberg et al., Proc. Natl. Acad. Sci. USA,92:3439-3443 (1995)). Candidate agents can be used in these animalmodels to determine if a candidate agent decreases one or more of thesymptoms associated with the inflammation.

Candidate agents can also be evaluated by directly testing theireffectiveness as MD2-antagonist. For example, an ELISA can be used tocharacterize binding to MD2. Other suitable methods for characterizingMD2 antagonist activity are further described in the examples providedherein.

The methods of the present invention can be used to provide prophylacticand/or therapeutic treatment. MD2-antagonists can, for example, beadministered prophylactically to a subject in advance of the occurrenceof the development of HMGB1-mediated inflammation. Prophylactic (i.e.,preventive) administration is effective to decrease the likelihood ofthe subsequent occurrence of HMGB1-mediated inflammation in a subject,or decrease the severity of HMGB1-mediated inflammation thatsubsequently occurs. Prophylactic treatment may be provided to a subjectthat is at elevated risk of developing HMGB1-mediated inflammation, suchas a subject with a family history of HMGB1-mediated inflammation.

Alternatively, the compounds of the invention can be administeredtherapeutically to a subject that is already afflicted by HMGB1-mediatedinflammation. In such methods, the MD2-antagonist is administered afterthe onset of inflammation or infection. In one embodiment of therapeuticadministration, administration of the compounds is effective toeliminate the HMGB1-mediated inflammation; in another embodiment,administration of the compounds is effective to decrease the severity ofthe HMGB1-mediated inflammation or lengthen the lifespan of the subjectso afflicted.

The methods of the invention include administering an MD2-antagonist toa subject in need thereof. The subject is preferably a mammal, such as adomesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog,cat). More preferably, the subject is a human A subject can becharacterized as being in need if they appear to be suffering fromHMGB1-mediated inflammation. In some embodiments, the subject appears tohave HMGB1-mediated inflammation as the result of a diagnosis.

When treating HMGB1-mediated inflammation by administering anMD2-antagonist, it can also be useful to administer one or moreadditional compounds to treat the inflammation or cause of inflammation.For example, when treating inflammation due to infection, antimicrobialagents such as antiviral or antibacterial agents can be co-administeredto the subject.

In some embodiments, an antiviral agent is also administered to thesubject. The choice of antiviral agent will vary depending on thespecific virus and the severity of the patient's condition. Examples ofantiviral agents include abacavir, acyclovir, adefovir, amantadine,amprenavir, ampligen, arbidol, atazanavir, atripla, balavir,boceprevirertet, cidofovir, combivir, dolutegravir, darunavir,delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine,enfuvirtide, entecavir, famciclovir, fomivirsen, fosamprenavir,foscarnet, fosfonet, ganciclovir, ibacitabine, imunovir, idoxuridine,imiquimod, indinavir, inosine, interferon types I-III, lamivudine,lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir,nevirapine, nexavir, oseltamivir (Tamiflu), peginterferon α-2a,penciclovir, peramivir, PF-429242, pleconaril, podophyllotoxin,raltegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir,sofosbuvir, stavudine, tea tree oil, telaprevir, tenofovir, tenofovirdisoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada,traporved, valaciclovir (Valtrex), valganciclovir, vicriviroc,vidarabine, viramidine, zalcitabine, zanamivir (Relenza), andzidovudine.

In some embodiments, an antibacterial agent can be co-administered tothe subject. The choice of antibacterial agent will vary depending onthe specific bacteria and the severity of the patient's condition.Examples of antibacterial agents include antibacterial agents, such asquinolones, e.g., ciprofloxacin, ofloxacin, moxifloxacin,methoxyfloxacin, pefloxacin, norfloxacin, sparfloxacin, temafloxacin,levofloxacin, lomefloxacin, and cinoxacin; penicillins, e.g.,cloxacillin, benzylpenicillin, and phenylmethoxypenicillin;aminoglycosides, e.g., erythromycin and other macrolides; andantitubercular agents, such as rifampicin and rifapentine.

Administration and Formulation

The one or more compounds (e.g., MD2 antagonist) can be administered aspharmaceutically acceptable salts. Pharmaceutically acceptable saltrefers to the relatively non-toxic, inorganic and organic acid additionsalts of the compounds. These salts can be prepared in situ during thefinal isolation and purification of the compound, or by separatelyreacting a purified compound with a suitable counterion, depending onthe nature of the compound, and isolating the salt thus formed.Representative counterions include the chloride, bromide, nitrate,ammonium, sulfate, tosylate, phosphate, tartrate, ethylenediamine, andmaleate salts, and the like. See for example Haynes et al., J. Pharm.Sci., 94, p. 2111-2120 (2005).

Pharmaceutical compositions of the invention include an MD2 antagonisttogether with one or more of a variety of pharmaceutically acceptablecarriers for delivery to a subject, including a variety of diluents orexcipients known to those of ordinary skill in the art. For example, forparenteral administration, isotonic saline is preferred. For topicaladministration, a cream, including a carrier such as dimethylsulfoxide(DMSO), or other agents typically found in topical creams that do notblock or inhibit activity of the peptide, can be used. Other suitablecarriers include, but are not limited to, alcohol, phosphate bufferedsaline, and other balanced salt solutions.

The formulations may be conveniently presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Preferably, such methods include the step of bringing the active agentinto association with a carrier that constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing the active agent into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product into the desired formulations. Themethods of the invention include administering to a subject, preferablya mammal, and more preferably a human, the composition of the inventionin an amount effective to produce the desired effect. The MD2 antagonistcan be administered as a single dose or in multiple doses. Usefuldosages of the MD2 antagonist can be determined by comparing their invitro activity and their in vivo activity in animal models. Methods forextrapolation of effective dosages in mice, and other animals, to humansare known in the art; for example, see U.S. Pat. No. 4,938,949.

MD2 antagonists are preferably formulated in pharmaceutical compositionsand then, in accordance with the methods of the invention, administeredto a subject, such as a human patient, in a variety of forms adapted tothe chosen route of administration. The formulations include, but arenot limited to, those suitable for oral, inhaled, rectal, vaginal,topical, nasal, ophthalmic, or parenteral (including subcutaneous,intramuscular, intraperitoneal, and intravenous) administration.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as tablets, troches, capsules,lozenges, wafers, or cachets, each containing a predetermined amount ofthe active agent as a powder or granules, as liposomes containing theactive compound, or as a solution or suspension in an aqueous liquor ornon-aqueous liquid such as a syrup, an elixir, an emulsion, or adraught. Such compositions and preparations typically contain at leastabout 0.1 wt-% of the active agent. The amount of the MD2 antagonist issuch that the dosage level will be effective to produce the desiredresult in the subject.

Inhaled formulations include those designed for administration from aninhaler device. Compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, aerosols, and powders.Preferably, the compositions are administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Solution, suspension, or powder compositions may beadministered, preferably orally or nasally, from devices that deliverthe formulation in an appropriate manner Nasal spray formulationsinclude purified aqueous solutions of the active agent with preservativeagents and isotonic agents. Such formulations are preferably adjusted toa pH and isotonic state compatible with the nasal mucous membranes.

Formulations for rectal or vaginal administration may be presented as asuppository with a suitable carrier such as cocoa butter, orhydrogenated fats or hydrogenated fatty carboxylic acids. Ophthalmicformulations are prepared by a similar method to the nasal spray, exceptthat the pH and isotonic factors are preferably adjusted to match thatof the eye. Topical formulations include the active agent dissolved orsuspended in one or more media such as mineral oil, petroleum,polyhydroxy alcohols, or other bases used for topical pharmaceuticalformulations.

The tablets, troches, pills, capsules, and the like may also contain oneor more of the following: a binder such as gum tragacanth, acacia, cornstarch or gelatin; an excipient such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acid,and the like; a lubricant such as magnesium stearate; a sweetening agentsuch as sucrose, fructose, lactose, or aspartame; and a natural orartificial flavoring agent. When the unit dosage form is a capsule, itmay further contain a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials may be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules may be coated with gelatin,wax, shellac, sugar, and the like. A syrup or elixir may contain one ormore of a sweetening agent, a preservative such as methyl- orpropylparaben, an agent to retard crystallization of the sugar, an agentto increase the solubility of any other ingredient, such as a polyhydricalcohol, for example glycerol or sorbitol, a dye, and flavoring agent.The material used in preparing any unit dosage form is substantiallynontoxic in the amounts employed. The MD2 antagonist may also beincorporated into sustained-release preparations and devices.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1: MD2 is Required for Disulfide HMGB1-Dependent TLR4Signaling

Innate immune receptors for pathogen- and damage-associated molecularpatterns (PAMP and DAMP) orchestrate inflammatory responses to infectionand injury. Secreted by activated immune cells or passively released bydamaged cells, HMGB1 is subjected to redox modification that distinctlyinfluences its extracellular functions. Previously, it was unknown howthe TLR4 signalosome distinguished between HMGB1 isoforms. Myeloiddifferentiation factor 2 (MD2) carries a hydrophobic pocket folded bytwo anti-parallel β-sheets for binding LPS, and confers molecularspecificity for LPS interaction and TLR4 signaling. Meng et al., J BiolChem 285:8695-8702 (2010). Accordingly, here we reasoned that MD2 maysimilarly discriminate different HMGB1 isoforms to facilitateTLR4-dependent signaling.

Here we demonstrate that the extracellular TLR4 adaptor MD2 bindsspecifically to the cytokine-inducing disulfide isoform of HMGB1, to theexclusion of other isoforms. Using MD2 deficient mice, as well as MD2silencing in macrophages, we show a requirement for HMGB1-dependent TLR4signaling. By screening HMGB1 peptide libraries, we identified atetramer (FSSE, designated P5779) as a specific MD2-antagonistpreventing MD2/HMGB1 interaction and TLR4 signaling. P5779 does notinterfere with LPS-induced cytokine/chemokine production, thuspreserving PAMP-mediated TLR4/MD2 responses. Furthermore, P5779 canprotect mice against hepatic ischemia/reperfusion injury, chemicaltoxicity and sepsis. These findings reveal a novel mechanism by whichinnate systems selectively recognize specific HMGB1 isoforms. Theresults may direct towards strategies aimed at attenuating DAMP-mediatedinflammation while preserving anti-microbial immune responsiveness.

Results and Discussion Cytokine-Inducing (Disulfide) HMGB1 EffectivelyBinds to MD2

HMGB1 contains three redox-sensitive cysteine residues that are modifiedby redox reactions to produce multiple HMGB1 isoforms thatextracellularly express or lack chemokine or cytokine activities. Toelucidate the underlying molecular mechanisms, we examined whether MD2,an extracellular adaptor receptor of the TLR4 signalosome, candiscriminate various HMGB1 isoforms with their distinct inflammatoryproperties. Different forms of HMGB1, produced by point mutations orchemical modifications by exposure to mercury thiolates or the reducingagent dithiothreitol, were tested for their MD2-binding properties.Consistent with previous reports (Venereau et al., J Exp Med209:1519-1528 (2012); Yang et al., Mol Med 18:250-259 (2012)), only thedisulfide HMGB1 isoform induced TNF secretion (FIG. 1A). Biosensor-basedsurface plasmon resonance analysis (BIAcore) confirmed that only thedisulfide HMGB1 binds to MD2 with high affinity (apparent Kd=12 nM)regardless whether MD2 or HMGB1 was immobilized on the sensor chip (FIG.1B). In contrast, HMGB1 was incapable of directly binding to TLR4 (FIG.1B) in the absence of MD2 although TLR4 was functionally active in MD2binding in BIAcore analysis, implicating MD2 as an essential participantin the HMGB1/TLR4 signaling pathway. Unlike the disulfide isoform ofHMGB1, H2S-modified, fully reduced or sulfonyl HMGB1 failed to induceTNF release from macrophage cultures (FIG. 1A), with more than a1,000-fold reduction in MD2 binding as compared to disulfide HMGB1 (FIG.1B). Notably, chemical modification of the cysteine 106 of the disulfideHMGB1 also abolished the TNF-stimulating and MD2-binding properties,indicating a critical role of the thiol-cysteine 106 in the regulationof HMGB1 cytokine activity (FIG. 1A-B).

To further study HMGB1-MD2 interactions, immune-precipitation assayswere employed to pull-down MD2 from HMGB1-expressing cell lysates.Co-incubation with calmodulin binding protein (CBP)-tagged disulfideHMGB1, but not CBP tag alone, pulled down MD2 protein from yeast cellstransfected with an MD2-expressing construct (FIG. 1C), confirming thatMD2 binds disulfide HMGB1. Furthermore, this interaction was blocked bymonoclonal anti-HMGB1 antibodies, but not by irrelevant IgG,demonstrating that the HMGB1-MD2 interaction is specific and targetableby antagonists (FIG. 1D).

MD2 is Required for HMGB1-Mediated Inflammatory Responses

To further assess the importance of MD2 in HMGB1-mediated cytokineinduction, we used siRNA to knockdown MD2 expression in murinemacrophage-like RAW 264.7 cells or human (THP-1) monocytes. Thesilencing of MD2 expression (by 80-90%) was accompanied by a significantreduction of HMGB1-stimulated NF-κB activation and TNF release in bothmurine macrophages and human monocytes (FIG. 2A). To confirm therequirement for MD2 in HMGB1-induced innate immune activation,thioglycollate-elicited peritoneal macrophages were isolated from wildtype and MD2 knockout (KO) mice, and stimulated by disulfide HMGB1.Disruption of MD2 expression resulted in complete impairment of bothLPS- and HMGB1-induced activation of NF-κB, secretion of cytokines (TNFand IL-6) and chemokines (e.g., RANTES and MCP-1) (FIG. 2B). The releaseof IL-12/p40 stimulated with HMGB1 is via an MD2 independent mechanism,likely attributable to signaling via other receptors.

HMGB1 is an important mediator of acetaminophen (APAP)-inducedhepatotoxicity. Antoine et al., J Hepatol 56:1070-1079 (2012). Toevaluate the in vivo importance of MD2 in HMGB1-induced inflammatoryresponses, we studied the impact of MD2 deficiency on sterileinflammation using the APAP intoxication model. The disruption of MD2expression resulted in a significant reduction in acute hepatic injury,as assessed by liver enzyme release (GLDH, AST and ALT) and histologicalanalysis of liver necrotic lesions compared to wild type (WT) micesubjected to APAP injection (FIG. 2C, arrow). Furthermore, the lessenedhepatic damage in MD2 KO mice was accompanied by significant reductionin cytokine (TNF and IL-6) release and APAP-induced animal lethality,confirming an essential role for MD2 in sterile inflammation and injury(FIG. 2C). Notably, serum HMGB1 levels were comparably elevated in wildtype and MD2 KO mice at 24 hour post APAP administration (FIG. 2C). Thecentral role of HMGB1 in APAP-induced liver toxicity was furtherconfirmed by using a HMGB1-neutralizing monoclonal antibody, whichsignificantly inhibited APAP-induced release of hepatic enzymes (ALT)and pro-inflammatory cytokines (TNF and IL-6), and improved survival(FIG. 3). Taken together, these in vivo experimental data reveal anessential role for MD2 and HMGB1 in the pathogenesis of sterile injury.

Development of a Novel MD2-Binding Peptide as an HMGB1-SpecificInhibitor

Having identified a critical involvement of the cysteine106 region ofHMGB1 in HMGB1/MD2 interaction and HMGB1/TLR4 signaling, we utilized arational strategy to screen for mimetic peptide inhibitors. A series oftrimer and tetramer peptides spanning the cysteine106 region andincorporating cysteine homologs were screened for MD2 binding propertiesusing the BIAcore technology and molecular docking technique (FIG.4A,C-D). Although most peptides lacked MD2-binding capacity, weidentified one epitope within the HMGB1 B box domain that acted as apotent HMGB1-specific inhibitor. Molecular docking simulation revealedthat the FSSE (P5779) tetramer fully extended into the hydrophobicpocket of MD2, thereby forming maximal van der Waals interaction withsurrounding hydrophobic residues along with an additional hydrogen bondwith the Tyr102 (FIG. 4D). Consequently, it bound to MD2 with a Kd valueof 0.65 μM and significantly inhibited HMGB1-induced TNF release fromhuman macrophages (FIG. 4A-B). This interaction was specific, as P5779failed to bind to other proteins such as HMGB1 and TLR4 in the absenceof MD2 (FIG. 4C). Similarly, scrambling the amino acid sequence of P5779(control peptide) abolished the MD2-binding capacity in BIAcoreexperiments (FIG. 4C) and in molecular docking analysis (FIG. 4D).

To evaluate the therapeutic potential of the MD2-binding peptide, wenext studied whether P5779 was capable of disrupting MD2/HMGB1interactions, thereby inhibiting HMGB1-induced cytokine production.P5779 inhibited the MD2/HMGB1 interaction in a concentration-dependentmanner when either MD2 or HMGB1 was coated onto the BIAcore sensor chip(FIG. 5A). Furthermore, P5779 inhibited HMGB1-induced TNF release inprimary human macrophages in a concentration-dependent fashion (FIG.5B). The effective concentration of P5779 that suppressed 50% TNFrelease (IC50) was approximately 5 μg/ml in the presence of HMGB1 at 1μg/ml. Scrambling the amino acid sequence of P5779 abolished thecapacity to inhibit HMGB1-induced TNF release (FIG. 5B). Exposure ofmacrophages to P5779 failed to inhibit TNF release mediated by Poly I:C,S100A12, LPS, PGN and CpG DNA (FIG. 5B). P5779 also significantlyreduced HMGB1-induced release of other cytokines including IL-6 andIL-12p40/p70 and chemokines such as RANTES and MCP-1 (FIG. 5C). P5779did not inhibit LPS-stimulated cytokine/chemokine release in vitro inmacrophages (FIG. 5D), and failed to attenuate LPS-induced systemiccytokine levels in vivo, even when administered at high doses (8 mg/kg)in mice (FIG. 5E). Thus, P5779 selectively attenuates HMGB1-MD2-TLR4signaling without inhibiting macrophages activation in response toPAMPs.

Therapeutic Efficacy of MD2-Targeted P5779 in Acetaminophen (APAP)Toxicity, Ischemia and Sepsis

In the APAP-induced liver toxicity model, P5779 treatmentdose-dependently reduced APAP-induced elevation of hepatic serum enzymes(AST, ALT), pro-inflammatory cytokines (TNF), liver necrosis andimproved survival (FIG. 6A, arrow). In sterile injury mediated byhepatic ischemia/reperfusion (I/R), P5779 also significantly bluntedhepatic serum enzyme release (AST, ALT) and neutrophil infiltration(FIG. 6B, arrow). In addition, treatment with P5779 in a sepsis modelinduced by cecal ligation and puncture (CLP) significantly anddose-dependently improved survival rates as compared to scrambledpeptide-treated controls (FIG. 6C). Importantly, P5779 was effectiveeven when administered 24 hours after onset of the peritonitis,consistent with the known delayed pathogenic role of HMGB1 in sepsissequelae. Taken together, these results indicate that P5779 disruptsbinding of disulfide HMGB1 to MD2 thereby attenuating HMGB1-mediatedorgan failure and mortality in vivo.

These results reveal a novel mechanism of selective innate immune cellrecognition of HMGB1 by MD2 that discriminates the HMGB1 isoforms. Byscreening HMGB1 peptide libraries, we identified a novel tetramerpeptide (FSSE, P5779) that specifically prevents the MD2-HMGB1interaction without impairing the MD2/LPS/TLR4 signaling in innateimmune cells. This peptide conferred protection not only in animalmodels of sterile injury-elicited inflammatory diseases but alsofollowing a lethal infection challenge, opening the possibility ofdeveloping novel therapeutic strategies to attenuate DAMP-mediatedinjurious inflammatory responses without inhibiting PAMP-elicited innateimmunity.

MD2 carries a β-cup fold structure composed of two anti-parallelβ-sheets that form a large hydrophobic pocket for binding to LPS. Theestimated affinity of MD2 binding to HMGB1 (12 nM) is comparable to MD2binding to LPS (65 nM) (Visintin et al., J Immunol., 175(10):6465-722005). Further structural analysis is required to reveal the disulfideHMGB1 binding site on MD2.

HMGB1-neutralizing antibodies are protective against sterile injury(Tsung et al., J Exp Med 201:1135-1143, 2005), and agents capable ofinhibiting HMGB1 release or its extracellular activities (Wang et al.,Science 285:248-251 (1999)) also confer protection against sepsis.During early stages of sepsis, PAMP-mediated inflammatory responses areessential to host defense. At later stages, the release of DAMPsamplifies the cytokine storm and organ dysfunction. Wang et al., ExpertOpin Ther Targets 18:257-268 (2014). This notion is supported by recentobservations that HMGB1 levels are persistently elevated during laterstages of sepsis, despite termination of the initial infection, andcontribute to long-term pathological consequences of sepsis.Valdes-Ferrer et al., Shock 40:492-495 (2013). Microbial-induced sepsiscan be clinically indistinguishable from the sterile injury-elicitedsystemic inflammatory response syndrome (SIRS). Sursal et al., Shock39:55-62 (2013). Based on the findings that TLR4/MD2 acts as a mutuallyexclusive signaling receptor complex for disulfide HMGB1, it is possibleto develop strategies that selectively attenuate DAMP-mediatedinflammatory responses while preserving PAMP-mediated signaling.

Substantial evidence supports the necessity to preserve earlyPAMP-mediated innate immune responses to counteract microbialinfections. For instance, defective TLR4 signaling in C3H/HeJ mice isassociated with aggravated disease severity and increased mortality inanimal models of infection. Khanolkar et al., J Virol 83:8946-8956(2009). LPS enhances macrophage phagocytic activity through TLR4 andselective deletion of TLR4 on myeloid cells impairs bacterial clearancein the CLP model. Deng et al., J Immunol 190:5152-5160 (2013). Thesefindings emphasize the importance of generating therapeutic approachesto selectively target damage-mediated inflammation while preservingphysiological protective immune responses. The discovery of P5779 as anMD2-targeting selective inhibitor for the DAMP-, but not thePAMP-elicited inflammatory responses provides such a novel therapeutictool.

Materials and Methods

Reagents. Human TLR4/MD2 complex, human MD2, TLR2, soluble RAGE wereobtained from R & D system Inc., (Minneapolis, Minn.).Lipopolysaccharide (LPS, E. coli. 0111:B4), acetaminophen, triton X-114,peptidoglycan from Bacillus subtili, blasticidin S., NaSH, mouse IgG,human macrophage-colony stimulating factor (M-CSF) were purchased fromSigma (St. Louis, Mo.). Protein A/G agarose andisopropyl-D-thiogalactopyranoside (IPTG) were from Pierce (Rockford,Ill.). NHS-activated sepharose 4 fast flow beads were obtained from GEHealthcare (Cat #17-0906-01, Uppsala, Sweden). Thioglycollate medium waspurchased from Becton Dickinson Co., (Sparks, Md.). Ultra pure LPS (Cat#tlrl-pelps), polyinosinic-polycytidylic acid (poly I:C) and type B CpGoligo-nucleotide were obtained from InVivogen (San Diego, Calif.). HumanS100 A12 was from Circulex Co. (Bangkok, Thailand). Anti-human and mouseMD2 antibodies were obtained from Imgenex (San Diego, Calif.). Anti-CBPtag antibody was from GenScript (Piscataway, N.J.). Anti-p50 antibody(E381) and anti-p65 antibody (sc-372) were obtained from Epitomics(Burlingame, Calif.) and Santa Cruz Biotech (Dallas, Tex.),respectively. Serum ALT and AST levels were determined by color endpointassay kits from BIOO Scientific (Austin, Tex.).

Preparations of HMGB1 proteins, antibodies and peptides. RecombinantHMGB1 was expressed in E. coli and purified to homogeneity as describedpreviously. Li et al., J Immunol Methods 289:211-223 (2004). Thiscytokine-stimulating HMGB1 contains a disulfide bond between cysteines23 and 45, and reduced thiol on cysteine 106, characterized by liquidchromatography tandem mass spectrometric analysis (LC-MS/MS). Yang etal., Mol Med 18:250-259 (2012). HMGB1 with redox modifications wascreated chemically by a synthetic formation of mercury thiolate oncysteine at position 106 (Hg-HMGB1), by S-sulfhydration (H2S) to convertcysteine thiol (—SH) group to —SSH or by mutation of cysteine 106 toalanine (C106A HMGB1) as described previously. Yang et al., Proc NatlAcad Sci USA 107:11942-11947 (2010). HMGB1 with cysteine modified by H2Swas generated by incubating HMGB1 with NaSH (5 mM) for 3 hours at roomtemperature. Oxidized or DTT-reduced HMGB1 was prepared as previouslydescribed (Yang et al., 2012). The LPS content in HMGB1 was measured bythe Chromogenic Limulus Amebocyte Lysate Assay (Lonza, Walkersville,Md.). HMGB1 was extracted with triton X-114 to remove any contaminatingLPS as described previously (Li et al., 2004). The purity and integrityof all recombinant proteins were verified by Coomassie Blue stainingafter SDS-PAGE, with a purity predominantly above 85%. The LPS contentin all HMGB1 protein preparations is non-detectable or less than 10pg/mg protein as measured by Limulus assay. Monoclonal anti-HMGB1antibody (2g7) was generated as reported previously. Qin et al., J ExpMed 203:1637-1642 (2006). Trimer or tetramer peptides (FSSE, FSSEY,FEEE, FEED, SSE, SFSE) and calmodulin binding peptide (CBP) were allcustomer-made from GeneMed Inc., (San Antonio, Tex.). The peptides werepurified to 90% purity as determined by HPLC. Endotoxin was notdetectable in the synthetic peptide preparations as measured by Limulusassay. The peptides were first dissolved in DMSO and further diluted inPBS as instructed by the manufacturer, and prepared freshly before use.Pre-casted mini-protean Tris-Tricine gels were from BioRad Lab(Hercules, Calif.).

Cell isolation and culture. Thioglycollate-ellicited peritonealmacrophages were obtained from mice (C57BL/6 or gene knock out, male,10-12 weeks old) injected with 2 ml of sterile 4% thioglycollate brothintraperitoneally as previously described (Yang et al., 2010). Murinemacrophage-like RAW 264.7 (TIB-71) and human leukemia monocytes THP-1(TIB-202) were obtained from American Type Culture Collection (ATCC,Rockville, Md.). Human primary monocytes were purified by densitygradient centrifugation through Ficoll from blood donated by normalindividuals as reported before (Yang et al., 2010). Human primarymacrophages in 96 well plate were stimulated with HMGB1 (1 μg/ml), TLR4agonist LPS at 4 ng/ml, TLR3 agonist poly I:C at 50 μg/ml, TLR2 agonistpeptidoglycan (PGN) at 5 μg/ml, RAGE agonist S100A12 at 50 μg/ml andTLR9 agonist CpG-DNA at 1 μM, plus increasing amounts of P5779 (orscrambled control peptide) as indicated for 16 hours. TNF released wasmeasured by ELISA.

Immuno-precipitation assay. Recombinant rat HMGB1 with a calmodulinbinding protein (CBP) tag, or CBP peptide alone (10 μg), was incubatedovernight with human MD2 supernatant (50 pre-cleared with calmodulinbeads) at 4° C. with gentle shaking. Human MD2 supernatant was obtainedfrom sf9 insect cells transfected with human MD2. Teghanemt et al.,J.B.C., 283:21881-21889 (2008). Both HMGB1 and MD2 supernatant containednon-detectable amounts of LPS as measured by Limulus amebocyte lysateassay. The mixture of CBP-HMGB1, or CBP and MD2 was then incubated withcalmodulin beads (30 μl drained beads) for 1 hour at 4° C. Afterextensive washing with PBS containing 0.1% triton X100, proteins boundto the beads were analyzed by Western blot probed with anti-human MD2 oranti-CBP antibodies.

Cytokine and NF-κB measurements. Levels of TNF and IL-6 released in thecell culture or from mice serum were measured by ELISA kits (R & DSystem Inc., Minneapolis, Minn.). Serum HMGB1 levels were measured byELISA kit (IBL international, Hamberg, Germany) Cytokine expressionprofile from thioglycollate-elicited peritoneal macrophages of mice orprimary human macrophages was determined by mouse or human cytokinearray C1 (Raybiotech, Norcross, Ga.) according to manufacturer'sinstructions. Twenty two cytokines or chemokines were determinedsimultaneously. NF-κB activation was analyzed by detecting p50 and p65expression in the nuclear fraction by western blot, ß-actin expressionwas also measured as control for equal loading of samples. Western blotswere scanned with a silver image scanner (Silver-scanner II, LacieLimited, Beaverton, Oreg.), and the relative band intensity wasquantified using ImageJ software (v1.59, National Institute of Health)and is expressed as a ratio to the amount of ß-actin.

Surface plasmon resonance analysis. Biacore T200 instrument was used forreal-time binding interaction studies. For HMGB1-MD2 binding analyses,human MD2 was immobilized onto a CMS series chip (GE Life Sciences). Oneflow-cell was used as a reference and thus immediately blocked uponactivation by 1 M ethanolamine (pH 8.5). The sample flow-cell wasinjected with disulfide HMGB1 (or isoforms) (in 10 mM acetate buffer, pH5.2) at a flow rate of 10 μL/min for 7 min at 25 C. Increasingconcentrations of disulfide HMGB1 or isoforms of HMGB1 (C106A, sulfonyl,fully reduced, Hg or H₂S-modified HMGB1, at 1 μM) were flowed overimmobilized MD2. In reverse fashion, HMGB1 was coated on the chip andvarious amounts of MD2 were added as analyte. Findings were confirmed byusing two additional human MD2 proteins from Dr. D. Golenbock(Worcester, Mass.) and Dr. Timothy Billiar (Pittsburgh, Pa.). ForTLR4-HMGB1 binding experiment, human TLR4 was coated on the chip anddisulfide HMGB1 (100 nM) was added as analyte. For peptide screeningexperiments, human MD2 was coated on the sensor chip, and various smallpeptides (FSSE (SEQ ID NO: 1), FSSEY (SEQ ID NO: 2), FEEE (SEQ ID NO:3), FEED (SEQ ID NO: 4), SSE, SFSE (SEQ ID NO: 5), 100 nM) were added asanalytes. The dissociation time was set for two minutes, followed by aone-minute regeneration using a 10 mM NaOH solution. The Kd wasevaluated using the BIAcore evaluation software. For experiments usingHMGB1 antibody to block MD2-HMGB1 interaction, human MD2 was coated onthe chip, HMGB1 was added as analyte (100 nM) plus increasing amounts ofHMGB1 mAb or control IgG and response units were recorded.

Molecular docking of MD2 with peptides. The crystal structure of theMD2/TLR4 was obtained from the Protein Data Base (PDB, code: 3VQ2), andmolecular docking was performed by using the MOE software as previouslydescribed. Zan et al., Mol Sim 6, 498-508 (2012). A molecularvisualization system, the Pymol 0.99, was used to construct the3-dimensional figures.

Knockdown MD2 in RAW 264.7 and THP-1 cells using siRNA. For MD2knockdown in RAW 264.7 cells, cells were transfected with mouse MD2 orcontrol siRNA (50 nM, on-target plus smart pool, Dharmacon, Lafayett,Colo.) using DharmaFect1 transfection reagent. To knockdown MD2 in THP-1cell, transfection with MD2 specific siRNA was performed by using AmaxaNucleofector kit. The efficiency of knockdown was confirmed by westernblot probed with anti-MD2 antibody at 48 hours after transfection. At 48hours post-transfection, cells were stimulated with HMGB1 (1 μg/ml) for16 hours. Cell lysate and supernatant were collected and analyzed bywestern blot or ELISA. NF-κB measurements on RAW 264.7, THP-1 or primarymouse macrophages from MD2 knockout mice were performed using NE-PERProtein Extraction Kit (Thermo Scientific, Hudson, N.H.).

Animals. Male C57BL/6 mice were obtained from Jackson Laboratory (BarHarbor, Me.). MD2 knockout (on C57BL/6 background) mice were purchasedfrom Riken Bio-Resource Center (Ibaraki, Japan). All animals weremaintained at The Feinstein Institute for Medical Research or Universityof Pittsburgh under standard temperature and light cycles, and allanimal procedures were approved by the institutional animal care and usecommittee.

For genotyping of MD2 KO mice from tail snips, PCR primers were designedby Riken Bio-Resource Center and were obtained from Invitrogen Inc.,(Carlsbad, Calif.). Same primers were used to identify wild type (PCRproduct=2,000 bp) and MD2 knockout (PCR product=800 bp) in genotyping.

For murine hepatic warm ischemia/reperfusion (I/R), a 70% warm liver I/RModel was performed as previously described. Tsung et al., J Exp Med201:1135-1143 (2005). Mice received intraperitoneal injection of P5779(500 μg/mouse) or vehicle at the time of surgery and were euthanized atsix hours afterwards. Whole blood was collected by cardiac puncture, andliver was harvested and fixed in 10% formalin for analysis.

For cecal ligation and puncture (CLP), C57BL/6 mice (male, 8-12 weeks ofage) were subjected to CLP procedure as described before (Yang et al.,2004). P5779 or scrambled control peptide were administeredintraperitoneally at 50 or 500 μg/mouse, treatment was given once a dayfor 4 days starting at 24 hours after CLP surgery. Survival wasmonitored for 2 weeks.

For the acetaminophen (APAP) hepatic toxicity model, three sets ofexperiments were conducted. In all experiments, mice were routinelyfasted overnight and received intraperitoneal (IP) injection of APAP(350 mg/kg for survival studies and 400 mg/kg for serum measurementswhen mice were euthanized at 24 h post-APAP) as previous described.Antoine et al., J Hepatol 56:1070-1079 (2012) The first set ofexperiments was performed using male MD2 KO or C57/BL6 mice (8-12 weeksold). Mice had APAP injection and were euthanized 24 hours later (forserum measurements) or monitored for 2 weeks (for survival studies). Thesecond set of experiments was performed using anti-HMGB1 antibody inAPAP model in wild type male (C57/BL6) mice. In survival experiments,mice had APAP injection and received anti-HMGB1 antibodies (5 μg/mouse,IP, once daily for 4 days followed by two additional doses once everyother day beginning at 2 hours post-APAP). Irrelevant non-immune IgG wasused as controls. For serum measurements, mice subjected to APAPinjection and received injection of monoclonal anti-HMGB1 antibody (5μg/mouse, injected IP at 2 and 7 hours post-APAP) and euthanized at 24hours post-APAP. The third set of experiments was used to assess theefficacy of P5779 in APAP model in wild type mice. Male C57BL/6 micereceived APAP injection plus P5779 (at 50 or 500 μg/mouse) or scrambledcontrol peptide (500 μg/mouse, i.p. injected at 2 and 7 hours post-APAP)and euthanized at 24 hours post-APAP. In survival experiments, mice hadinjection of APAP and received treatment of P5779 or control peptide(500 μg/mouse, i.p. once a day for 5 days starting at 2 hours post-APAP)and survival was monitored for 2 weeks. Hepatotoxicity was determined byserum levels of glutamate dehydrogenase (GLDH), alanineaminotransferease (ALT) and aspartate aminotransferase (AST) asdescribed previously. Antoine et al., Hepatology 58:777-787 (2013).

Histological evaluation. Harvested livers were fixed in 10% formalin,and embedded in paraffin. Five-μM sections were stained with hematoxylinand eosin. H&E staining of livers was performed by AML laboratory(Baltimore, Md.). The liver histology was evaluated in a blinded fashionand clinical scores were calculated based on the amount of necrosis andinflammation (cell swelling, loss of tissue structure, congestion) usinga previously reported method with modifications. Desmet et al., Journalof hepatology 38:382-386 (2003). Score 0=no evidence of necrosis orinflammation as assessed from three to four representative sections fromeach animal; 1=mild necrosis or inflammation <25% of the total areaexamined, 2=notable necrosis and inflammation (25-50% of the totalarea); 3=severe necrosis and inflammation (>50% area).

Statistical analysis. Data are presented as means+SEM unless otherwisestated. Differences between treatment groups were determined byStudent's t-test, one-way ANOVA followed by the least significantdifference test. Differences between groups in animal survival studieswere determined using two-tailed Fisher's exact test. Cytokine arraystudies were analyzed using the software UN-Scan-it from Silk scientificInc. (Orem, Utah). P values less than 0.05 are considered statisticallysignificant.

Statistics

The complete disclosure of all patents, patent applications, andpublications, and electronically available materials cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. In particular,while theories may be presented describing possible mechanisms throughwith the compounds are effective, the inventors are not bound bytheories described herein. The invention is not limited to the exactdetails shown and described, for variations obvious to one skilled inthe art will be included within the invention defined by the claims.

What is claimed is: 1-16. (canceled)
 17. A composition comprising apeptide having the amino acid sequence FSSE.
 18. The composition ofclaim 17, further comprising a pharmaceutically acceptable carrier. 19.The composition of claim 18, wherein the pharmaceutically acceptablecarrier provides a parenteral formulation.
 20. A method of treatinghigh-mobility group box protein B1 (HMGB1)-mediated inflammation in asubject, by administering a therapeutically effective amount of P5779 toa subject in need thereof, wherein P5779 is the tetrapeptide FSSE. 21.The method of claim 20, wherein the HMGB1-mediated inflammation iscaused by infection.
 22. The method of claim 21, wherein theHMGB1-mediated inflammation is caused by viral infection.
 23. The methodof claim 22, wherein the HMGB1-mediated inflammation is caused byinfluenza infection.
 24. The method of claim 20, wherein theHMGB1-mediated inflammation is caused by bacterial infection.
 25. Themethod of claim 20, wherein the method does not decrease anti-microbialimmune responsiveness.
 26. The method of claim 20, wherein the P5779 isadministered after the onset of infection.
 27. The method of claim 22,further comprising administering an antiviral agent to the subject. 28.The method of claim 20, wherein the HMGB1-mediated inflammation iscaused by sterile injury.
 29. The method of claim 20, wherein theHMGB1-mediated inflammation is caused by acetaminophen toxicity.
 30. Themethod of claim 20, wherein the subject is human.
 31. The method ofclaim 20, wherein the P5779 is administered in a pharmaceuticallyacceptable carrier.